title: Structural basis for the inhibition of bacterial multidrug exporters.
abstract: The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa. We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics. Drugs were shown to be exported by a functionally rotating mechanism through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π-π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa.
authors.affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Nakashima
authors.forename: Keisuke
authors.initials: K
authors.lastname: Sakurai
authors.forename: Seiji
authors.initials: S
authors.lastname: Yamasaki
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Hayashi
authors.forename: Chikahiro
authors.initials: C
authors.lastname: Nagata
authors.forename: Kazuki
authors.initials: K
authors.lastname: Hoshino
authors.forename: Yoshikuni
authors.initials: Y
authors.lastname: Onodera
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Nishino
authors.forename: Akihito
authors.initials: A
authors.lastname: Yamaguchi
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D055503:Protein Multimerization; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000378:metabolism*; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011744:Pyrimidinones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012399:Rotation
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011725:Pyridines; D011743:Pyrimidines; D011744:Pyrimidinones; C583203:ABI-PP compound; C083496:AcrB protein, E coli; C118597:MexB protein, Pseudomonas aeruginosa